Viral core protein-cationic lipid-nucleic acid-delivery complexes

ABSTRACT

A nucleic acid delivery complex is provided which comprises a condensed polypeptide/nucleic acid complex and a cationic lipid wherein the complex comprises (a) a nucleic acide sequence of interest (NOI); and (b) one or more viral nucleic acid packaging polypeptides, or derivatives thereof, said polypeptides or derivatives thereof being (i) capable of binding to the NOI; and (ii) capable of condensing the NOI; and wherein the NOI is heterologous to the polypeptide. Also provided is a method of introducing an NOI into a cell using the delivery vector.

FIELD OF THE INVENTION

[0001] The present invention relates to cationic lipid/protein/nucleicacid complexes comprising viral packaging proteins and their use in theefficient delivery of nucleic acids to cells, such as neuronal cells.

BACKGROUND OF THE INVENTION

[0002] Promising advances in non-viral gene transfer have been made as aresult of the production of synthetic liposomes formulated with cationiclipids that are able to transfect cells. However few of these complexeshave been examined for their ability to efficiently transfer DNA intoCNS cells and to obtain expression of a transgene. The ability totransfect neuronal cells efficiently and safely could provide a powerfultool for the elucidation of neuronal function and may lead to noveltreatments for neurological disorders.

[0003] Unfortunately, gene therapy for the CNS has been hampered by thelack of efficient means for transducing postmitotic neurons. Moststudies have utilized viral vectors for gene delivery. However, manyviral vectors are plagued by problems of immunity and cytotoxicity andare not easily manipulated by non-virologists ¹⁻³. Non-viral vectors arenow emerging as an alternative method of cellular transduction. The mostpromising advances in non-viral gene transfer have been in theproduction of synthetic liposomes formulated with cationic lipids(cytofectins) able to transfect cells. Such cationic liposomes arerelatively easy to use, have a broad applicability and lack cytotoxicity⁴.

[0004] Novel cationic liposome formulations are constantly beingdeveloped ⁵. However, few of these complexes have been examined fortheir ability to efficiently transduce cells within the CNS ⁶⁻⁹.Cationic liposomes act via electrostatic interactions with negativelycharged DNA and subsequently with cellular membranes where they aretaken across the cell membrane by a process of slow endocytosis^(6, 10, 11). They are frequently formulated using the neutral lipiddioleoyl-L-α-phosphatidylethanolamine (DOPE), which is extremelyefficient at endosomal buffering and disruption ^(8, 12). From theperinuclear space transfected genetic material is released from theliposome complex, transported to the nucleus and expressed. To date onlyliposomes formulated from N-[1-(2,3-dioleyloxy)propyl]-N,N,N trimethylammonium chloride (DOTMA) and DOPE, have been shown to mediatesuccessful transfection in the CNS ¹³⁻¹⁶. To be useful for gene therapyliposome complexes capable of transfecting CNS cells with highefficiency are needed.

[0005] A major limitation in non-viral mediated gene transfer is theformation of large aggregated molecules during the generation ofliposome:DNA complexes ⁵. These large aggregates may reduce theefficiency of transfection possibly by limiting endocytosis of thecomplexes. One approach to circumvent this is to reduce the size of DNAmolecules via DNA condensation prior to complex formation.Pre-condensation of DNA produces smaller complexes and improvedtransfection efficiencies ¹⁷⁻²³. Various polycations have beenidentified which are efficient at improving. liposome-mediatedtransfections. Of these, poly-L-lysine and protamine have produced themost dramatic results enabling increases of over 30 fold compared tocomplexes without pre-condensation in a variety of non-neuronal celllines ^(17, 21).

[0006] Protamine sulphate is particularly good at enhancing liposomaltransfection. Protamine is a naturally occurring polycation found in thehead of spermatozoa. The role of protamine is to condense DNA in spermand aid in its transfer to the egg nucleus. The nuclear targetingproperty of protamine makes it particularly attractive for genetransfer. Also, unlike the synthetic poly-L-lysine, which has a range oflarge molecular weights (18000-19200 Da), protamine is naturallyoccurring, smaller and more uniform in size (4000-4250 Da). Thesequalities mean there is less chance for immunogenic responses in thetarget tissue and the condensation is easier to control. Other naturallyoccurring DNA condensing proteins have also been used to enhancecationic liposome mediated DNA transfer. Fritz et al, ²² achievedapproximately 30 fold increases in lipofection using a recombinant humanH1 histone protein incorporating a nuclear localization signal (nls-H1).Also, the non-histone chromosomal high mobility group 1,2 protein hasbeen shown to improve lipofection and is used routinely in theHVJ-liposome method ^(20, 24).

SUMMARY OF THE INVENTION

[0007] We have examined viral-DNA associated proteins for their abilityto improve liposome based gene transfer. In particular we have comparedthe viral-coded synthetic peptide Mu1 and recombinant Vp1 protein ofadenoviras and polyomavirus respectively. Mu1 may play a role inadenoviral chromosome condensation while VP1 is the only structuralprotein of polyomavirus to exhibit DNA binding activity ²⁵-²⁷. Vp1, butnot Mu1 contains an embedded classical nuclear localization signal (NLS)similar to that found in HMG-1,2 and nls-H1 ²⁶. We found that Mu1, butnot Vp1, significantly improved cationic liposome mediated gene transferin cells derived from the nervous system and kidney. We also found thatMu1 enhancement was greater in differentiated cells indicating thepossible usefulness of this approach for neuronal cells in vivo.

[0008] These findings have implications for experimental and therapeuticuses of liposome-mediated delivery of DNA to CNS cells.

[0009] Accordingly, the present invention provides a non-viral nucleicacid delivery vector comprising a condensed polypeptide/nucleic acidcomplex and a cationic lipid, wherein the complex comprises

[0010] (a) a nucleic acid sequence of interest (NOI); and

[0011] (b) one or more viral nucleic acid packaging polypeptides, orderivatives thereof, said polypeptides or derivatives thereof being (i)capable of binding to the NOI; and (ii) capable of condensing the NOI;and wherein the NOI is heterologous to the polypeptide.

[0012] Preferably, at least one polypeptide is an adenoviral nucleicacid packaging polypeptide, or derivative thereof. More preferably, theadenoviral polypeptide is Mu1, pV or pVII or a derivative thereof.

[0013] The term “heterologous to the polypeptide” means that viral NOIsthat naturally occur in combination with the viral packaging polypeptideare excluded.

[0014] In a preferred embodiment, the vector further comprises apolypeptide comprising a nuclear localisation sequence (NLS). Morepreferably, the polypeptide comprising a nuclear localisation sequence(NLS) is adenoviral pV or a derivative thereof.

[0015] The present invention also provides a condensedpolypeptide/nucleic acid complex comprising a cationic lipid, apolypeptide component and a nucleic acid component, for use indelivering the nucleic acid component to a nucleus of a eukaryotic cell,wherein

[0016] (i) the polypeptide component is a viral nucleic acid packagingpolypeptide, or derivative thereof;

[0017] (ii) the polypeptide component or derivative thereof is capableof binding to the NOI; and

[0018] (iii) the polypeptide component or derivative thereof is capableof condensing the NOI; and wherein the nucleic acid is heterologous tothe polypeptide.

[0019] Preferably, at least one polypeptide is an adenoviral nucleicacid packaging polypeptide, or derivative thereof. More preferably, theadenoviral polypeptide is Mu1, pV or pVII or a derivative thereof.

[0020] In a preferred embodiment, the complex further comprises apolypeptide comprising a nuclear localisation sequence (NLS). Morepreferably, the polypeptide comprising a nuclear localisation sequence(NLS) is adenoviral pV or a derivative thereof.

[0021] The present invention also provides a method of producing anon-viral nucleic acid delivery vector comprising a condensedpolypeptide/ nucleic acid complex and a cationic lipid, which methodcomprises

[0022] (a) contacting an nucleic acid sequence of interest (NOI) with aviral nucleic acid packaging polypeptide or derivative thereof, saidpolypeptide component or derivative thereof being (i) capable of bindingto the NOI; and (ii) capable of condensing the NOI; and wherein the NOIis heterologous to the polypeptide; and

[0023] (b) contacting the nucleic acid/polypeptide complex thus formedwith a cationic lipid.

[0024] The present invention further provides a method of introducing anucleic acid sequence of interest (NOI) into a eukaryotic cell whichmethod comprises contacting the cell with a complex of the inventionwherein the complex comprises the NOI. Preferably the cell is aneuronal, cancer or epithelial cell.

[0025] In an alternative embodiment, a viral nucleic acid nuclearlocalisation/delivery polypeptide may be used instead of, or in additionto a viral nucleic acid packaging polypeptide. Indeed, some viralpolypeptides combine both functions.

DETAILED DESCRIPTION OF THE INVENTION

[0026] Although in general the techniques mentioned herein are wellknown in the art, reference may be made in particular to Sambrook etal., Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al.,Short Protocols in Molecular Biology (1999) 4^(th) Ed, John Wiley &Sons, Inc.

[0027] A. Polypeptide Components

[0028] 1. Viral Nucleic Acid Packaging Polypeptides

[0029] The term “viral nucleic acid packaging polypeptides” typicallyincludes polypeptides encoded by viral genomes that occur naturally inviral particles where their function is to package, in particularcondense, and deliver into the nucleus the nucleic acids constitutingthe viral genome into the virion. Also included are homologues andderivatives thereof, such as fragments, as discussed below.

[0030] Examples of viral nucleic acid packaging polypeptides includeviral core proteins such as hepatitis B core antigen and adenoviral coreproteins, Mu1, pV and pVII and their equivalents proteins in otheradenoviruses, such as Mastadenoviruses (mammalian adenoviruses) andAviadenoviruses, (bird adenoviruses). A particularly preferred viralnucleic acid packaging polypeptide for use in the present invention isthe Mu1 polypeptide shown immediately below as SEQ I.D. No. 1.

[0031]NH₂-Met-Arg-Arg-Ala-His-His-Arg-Arg-Arg-Arg-Ala-Ser-His-Arg-Arg-Met-Arg-Gly-Gly-OH(SEQ I.D. No. 1).

[0032] A viral nucleic acid packaging polypeptide for use in the presentinvention is capable of binding to nucleic acids, typically in anon-specific manner, preferably causing condensation of the nucleicacid. It is generally preferred that the condensed NOI has a size ofequal to or less than 200 nm, such as from 50 to 200 nm, for optimalefficiency of delivery to a target cell.

[0033] The ability of viral polypeptides to bind to nucleic acids may bedetermined in vitro using techniques such as gel electrophoresisincluding gel retardation assays (see materials and methods section andresults section) and electrophoretic band shift mobility assays,ethidium bromide exclusion assays and affinity chromatography (forexample using single- or double-stranded DNA cellulose).

[0034] The ability of viral polypeptides to condense nucleic acids maybe determined by, for example, circular dichroism (CD) spectroscopy(see, for example, Sato and Hosokawa, 1984, J. Biol. Chem. 95:1031-1039).

[0035] Generally the viral polypeptides, or homologues or derivativesthereof, will comprise a number of positively charged amino acidresidues at physiological pH (such as pH 7.4). Preferably the overallnet charge on the viral polypeptide is positive at physiological pH. Inparticular, it is preferred that the charge:amino acid ratio is at least+0.3, preferably at least +0.4, +0.5 or +0.6.

[0036] It is preferred that the viral polypeptides, or homologues orderivatives thereof comprise arginine residues rather than lysineresidues or a mixture of both. It is also particularly preferred thatthe viral polypeptides, or homologues or derivatives thereof compriseone or more histidine residues, preferably two or more histidineresidues. In addition, the viral polypeptides, or homologues orderivatives thereof will typically comprise a number of highlyhydrophobic residues, such as alanine, for example two or morehydrophobic residues.

[0037] It will be understood that amino acid sequences for use in theinvention are not limited to naturally occurring viral nucleic acidpackaging polypeptides but also include homologous sequences obtainedfrom any source, for example related viral/bacterial proteins, cellularhomologues and synthetic peptides, as well as variants or derivatives,such as fragments, thereof.

[0038] In the context of the present invention, a homologous sequence istaken to include an amino acid sequence which is at least 60, 70, 80 or90% identical, preferably at least 95 or 98% identical at the amino acidlevel over at least 10 preferably at least 20, 30, 40 or 50 amino acidswith a viral core polypeptide, for example the Mu1 sequence shown as SEQI.D. No. 1. In particular, homology should typically be considered withrespect to those regions of the sequence known to be essential fornucleic acid binding rather than non-essential neighbouring sequences.Although homology can also be considered in terms of similarity (i.e.amino acid residues having similar chemical properties/functions), inthe context of the present invention it is preferred to express homologyin terms of sequence identity.

[0039] Homology comparisons can be conducted by eye, or more usually,with the aid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homologybetween two or more sequences.

[0040] % homology may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment Typically, such ungapped alignments are performed only over arelatively short number of residues (for example less than 50 contiguousamino acids).

[0041] Although this is a very simple and consistent method, it fails totake into consideration that for example, in an otherwise identical pairof sequences, one insertion or deletion will cause the following aminoacid residues to be put out of alignment, thus potentially resulting ina large reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

[0042] However, these more complex methods assign “gap penalties” toeach gap that occurs in the alignment so that, for the same number ofidentical amino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example when using the GCG Wisconsin Bestfitpackage (see below) the default gap penalty for amino acid sequences is−12 for a gap and −4 for each extension.

[0043] Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software than can perform sequence comparisons include, but arenot limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter18), FASTA (Atschul et al, 1990, J. Mol. Biol., 403-410) and theGENEWORKS suite of comparison tools. Both BLAST and FASTA are availablefor offline and online searching (see Ausubel et al., 1999 ibid, pages7-58 to 7-60). However it is preferred to use the GCG Bestfit program.

[0044] Although the final % homology can be measured in terms ofidentity, the alignment process itself is typically not based on anall-or-nothing pair comparison. Instead, a scaled similarity scorematrix is generally used that assigns scores to each pairwise comparisonbased on chemical similarity or evolutionary distance. An example ofsuch a matrix commonly used is the BLOSUM62 matrix—the default matrixfor the BLAST suite of programs. GCG Wisconsin programs generally useeither the public default values or a custom symbol comparison table ifsupplied (see user manual for further details). It is preferred to usethe public default values for the GCG package, or in the case of othersoftware, the default matrix, such as BLOSUM62.

[0045] Once the software has produced an optimal alignment, it ispossible to calculate % homology, preferably % sequence identity. Thesoftware typically does this as part of the sequence comparison andgenerates a numerical result.

[0046] The terms “derivative” in relation to the amino acid sequencesused in the present invention includes any substitution of, variationof, modification of, replacement of, deletion of or addition of one (ormore) amino acids from or to the sequence providing the resultant aminoacid sequence has nucleic acid binding and condensation activity,preferably having at least the same activity as the unmodifiedpolypeptides.

[0047] Viral polypeptides may be modified for use in the presentinvention. Typically, modifications are made that maintain the nucleicacid binding and condensation properties of the sequence. Amino acidsubstitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30substitutions provided that the modified sequence retains nucleic acidbinding and condensation properties. Amino acid substitutions mayinclude the use of non-naturally occurring analogues, for example toincrease blood plasma half-life of a therapeutically administeredpolypeptide.

[0048] In particular, it may be desirable to make amino acidsubstitutions to increase the net positive charge, at physiological pH,of a naturally occurring viral packaging polypeptide. Positively chargedamino acids include arginine, lysine and histidine. Arginine is the mosthighly charged of the naturally occurring amino acids and isparticularly preferred. ALIPHATIC Non-polar GAP ILV Polar - unchargedCSTM NQ Polar - charged DE KR AROMATIC HFWY

[0049] Conservative substitutions may be made, for example according tothe Table above. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other:

[0050] Polypeptides for use in the invention may be made by recombinantmeans, for example as described below. However they may also be made bysynthetic means using techniques well known to skilled persons such assolid phase synthesis. Polypeptides for use in the invention may also beproduced as fusion proteins, for example to aid in extraction andpurification. Examples of fusion protein partners includeglutathione-S-transferase (GST), 6×His, GAL4 (DNA binding and/ortranscriptional activation domains) and β-galactosidase. It may also beconvenient to include a proteolytic cleavage site between the fusionprotein partner and the protein sequence of interest to allow removal offusion protein sequences. Preferably the fusion protein partner will nothinder the biological activity of the protein of interest sequence.

[0051] Polypeptides for use in the invention may be in a substantiallyisolated form. It will be understood that the polypeptides may be mixedwith carriers or diluents which will not interfere with the intendedpurpose of the polypeptides and still be regarded as substantiallyisolated. The polypeptides may also be in a substantially purified form,in which case generally more than 90%, e.g. 95%, 98% or 99% of theprotein in the preparation comprises polypeptides for use in theinvention.

[0052] 2. Polypeptides Comprising Nuclear Localisation Sequences

[0053] In a preferred embodiment, the delivery vector/complex of theinvention further comprises a polypeptide comprising a nuclearlocalisation sequence (NLS). In general, NLSs are well known in the art(see, for example, Dingwall and Laskey, 1991, Trends. Biochem. Sci. 16:478-481). However, it is particularly preferred to use the NLS ofadenovirus core protein pV. The NLS of pV has the sequenceRPRRRATTRRRTTTGTRRRRRRR (SEQ I.D. No. 2) corresponding to amino acids315-337 (D. Matthews, submitted.) A further NLS is present in theN-terminus (KPRKLKRVKKKKK—SEQ I.D. No. 3), although the C-terminal NLSis preferred.

[0054] The NLS may be present on a separate polypeptide molecule to thepackaging polypeptide or as part of the same polypeptide chain, forexample in a fusion protein.

[0055] B. Nucleic Acid Sequences of Interest

[0056] Nucleic acid sequences of interest (NOIs) intended to bedelivered to cells using the delivery vector or complex of the inventionmay comprise DNA or RNA. They may be single-stranded or double-stranded.They may also be polynucleotides which include within them synthetic ormodified nucleotides. A number of different types of modification tooligonucleotides are known in the art. These include methylphosphonateand phosphorothioate backbones, addition of acridine or polylysinechains at the 3′ and/or 5′ ends of the molecule. For the purposes of thepresent invention, it is to be understood that the polynucleotidesdescribed herein may be modified by any method available in the art Suchmodifications may be carried out in order to enhance the in vivoactivity or life span of the NOIs.

[0057] The NOI typically comprises a heterologous gene. The term“heterologous gene” encompasses any gene, The heterologous gene may beany allelic variant of a wild-type gene, or it may be a mutant gene. Theterm “gene” is intended to cover nucleic acid sequences which arecapable of being at least transcribed. Thus, sequences encoding mRNA,tRNA and rRNA, as well as antisense constructs, are included within thisdefinition. Nucleic acids may be, for example, ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or analogues thereof. Sequences encodingmRNA will optionally include some or all of 5′ and/or 3′ transcribed butuntranslated flanking sequences naturally, or otherwise, associated withthe translated coding sequence. It may optionally further include theassociated transcriptional control sequences normally associated withthe transcribed sequences, for example transcriptional stop signals,polyadenylation sites and downstream enhancer elements.

[0058] The transcribed sequence of the heterologous gene is preferablyoperably linked to a control sequence permitting expression of theheterologous gene in mammalian cells, preferably neuronal cells, such ascells of the central and peripheral nervous system, cancer or epithelialcells. The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequence.

[0059] The control sequence comprises a promoter allowing expression ofthe heterologous gene and a signal for termination of transcription. Thepromoter is selected from promoters which are functional in mammalian,preferably human cells. The promoter may be derived from promotersequences of eukaryotic genes. For example, it may be a promoter derivedfrom the genome of a cell in which expression of the heterologous geneis to occur, preferably a cell of the mammalian central or peripheralnervous system. With respect to eukaryotic promoters, they may bepromoters that function in a ubiquitous manner (such as promoters ofβ-actin, tubulin) or, alternatively, a tissue-specific manner (such aspromoters of the genes for pyruvate kinase). They may also be promotersthat respond to specific stimuli, for example promoters that bindsteroid hormone receptors. Viral promoters may also be used, for examplethe Moloney murine leukaemia virus long terminal repeat (MMLV LTR)promoter or promoters of herpes virus genes.

[0060] It may also be advantageous for the promoters to be inducible sothat the levels of expression of the heterologous gene can be regulatedduring the life-time of the cell. Inducible means that the levels ofexpression obtained using the promoter can be regulated.

[0061] In addition, any of these promoters may be modified by theaddition of further regulatory sequences, for example enhancersequences. Chimeric promoters may also be used comprising sequenceelements from two or more different promoters described above.Furthermore, the use of locus control regions (LCRs) may be desirable.

[0062] The heterologous gene will typically encode a polypeptide oftherapeutic use. In accordance with the present invention, suitable NOIsequences include those that are of therapeutic and/or diagnosticapplication such as, but are not limited to: sequences encodingcytokines, chemokines, hormones, antibodies, engineeredimmunoglobulin-like molecules, a single chain antibody, fusion proteins,enzymes, immune co-stimulatory molecules, immunomodulatory molecules,anti-sense RNA, a transdominant negative mutant of a target protein, atoxin, a conditional toxin, an antigen, a tumour suppressor protein andgrowth factors, membrane proteins, vasoactive proteins and peptides,anti-viral proteins and ribozymes, and derivatives therof (such as withan associated reporter group).

[0063] Examples of polypeptides of therapeutic use include neurotrophicfactors such as nerve growth factor (NGF), ciliary neurotrophic factor(CNTF), brain-derived neurotrophic factor (BNTF) and neurotrophins (suchas NT-3, NT-4/5) which have potential as therapeutic agents for thetreatment of neurological disorders such as Parkinson's disease.

[0064] Suitable NOIs for use in the present invention in the treatmentor prophylaxis of cancer include NOIs encoding proteins which: destroythe target cell (for example a ribosomal toxin), act as: tumoursuppressors (such as wild-type p53); activators of anti-tumour immunemechanisms (such as cytokines, co-stimulatory molecules andimmunoglobulins); inhibitors of angiogenesis; or which provide enhanceddrug sensitivity (such as pro-drug activation enzymes); indirectlystimulate destruction of target cell by natural effector cells (forexample, strong antigen to stimulate the immune system or convert aprecursor substance to a toxic substance which destroys the target cell(for example a prodrug activating enzyme). Encoded proteins could alsodestroy bystander tumour cells (for example with secreted antitumourantibody-ribosomal toxin fusion protein), indirectly stimulateddestruction of bystander tumour cells (for example cytokines tostimulate the immune system or procoagulant proteins causing localvascular occlusion) or convert a precursor substance to a toxicsubstance which destroys bystander tumour cells (eg an enzyme whichactivates a prodrug to a diffusible drug).

[0065] NOI(s) may be used which encode antisense transcripts orribozymes which interfere with the expression of cellular or pathogengenes, for example, with expression of cellular genes for tumourpersistence (for example against aberrant myc transcripts in Burkittslymphoma or against bcr-abl transcripts in chronic myeloid leukemia. Theuse of combinations of such NOIs is also envisaged.

[0066] Instead of, or as well as, being selectively expressed in targettissues, the NOI or NOIs may encode a pro-drug activation enzyme orenzymes which have no significant effect or no deleterious effect untilthe individual is treated with one or more pro-drugs upon which theenzyme or enzymes act. In the presence of the active NOI, treatment ofan individual with the appropriate pro-drug leads to enhanced reductionin tumour growth or survival.

[0067] A pro-drug activating enzyme may be delivered to a tumour sitefor the treatment of a cancer. In each case, a suitable pro-drug is usedin the treatment of the patient in combination with the appropriatepro-drug activating enzyme. An appropriate pro-drug is administered inconjunction with the vector. Examples of pro-drugs include: etoposidephosphate (with alkaline phosphatase); 5-fluorocytosine (with cytosinedeaminase); doxorubicin-N-p-hydroxyphenoxyacetamide (withpenicillin-V-amidase); para-N-bis(2-chloroethyl) aminobenzoyl glutamate(with carboxypeptidase G2); cephalosporin nitrogen mustard carbamates(with β-lactamase); SR4233 (with P450 Reducase); ganciclovir (with HSVthymidine kinase); mustard pro-drugs with nitroreductase andcyclophosphamide (with P450).

[0068] Examples of suitable pro-drug activation enzymes for use in theinvention include a thymidine phosphorylase which activates the5-fluoro-uracil pro-drugs capcetabine and furtulon; thymidine kinasefrom herpes simplex virus which activates ganciclovir, a cytochrome P450which activates a pro-drug such as cyclophosphamide to a DNA damagingagent; and cytosine deaminase which activates 5-fluorocytosine.Preferably, an enzyme of human origin is used

[0069] NOIs may also encode antigenic polypeptides for use as vaccines.Preferably such antigenic polypeptides are derived from pathogenicorganisms, for example bacteria or viruses. Examples of such antigenicpolypeptides include hepatitis C virus antigens, hepatitis B surface orcore antigens, HIV antigens, pertussis toxin, cholera toxin ordiphtheria toxin.

[0070] NOIs may also include marker genes (for example encodingβ-galactosidase or green fluorescent protein) or genes whose productsregulate the expression of other genes (for example, transcriptionalregulatory factors).

[0071] Where a disease is caused by a defective gene, NOIs may beadmistered that encode a fully functional allele of the gene, such as inthe case of cystic fibrosis. The molecular basis for a variety ofgenetic disorders has been identified and wild type functional sequencescloned. It may be desirable to include in the NOI flanking sequences tothe therapeutic gene that are homologous to the corresponding flankingsequences in the genome to allow for replacement of the defective geneby homologous recombination.

[0072] Gene therapy and other therapeutic applications may well requirethe administration of multiple genes. The expression of multiple genesmay be advantageous for the treatment of a variety of conditions. Sincethere is no limitation in the size of NOI that may be incorporated intoa delivery vector or complex of the invention, it should be possible totarget cells with multiple genes simultaneously.

[0073] C. Cationic Lipids

[0074] A variety of cationic lipids is known in the art—see for exampleWO95/02698, the disclosure of which is herein incorporated by reference,some of which is reproduced below. Example structures of cationic lipidsuseful in this invention are provided in Table 1 of WO95/02698.Generally, any cationic lipid, either monovalent or polyvalent, can beused in the compositions and methods of this invention. Polyvalentcationic lipids are generally preferred. Cationic lipids includesaturated and unsaturated allyl and alicyclic ethers and esters ofamines, amides or derivatives thereof. Straight-chain and branched alkyland alkene groups of cationic lipids can contain from 1 to about 25carbon atoms. Preferred straight-chain or branched alkyl or alkenegroups have six or more carbon atoms. Alicyclic groups can contain fromabout 6 to 30 carbon atoms. Preferred alicyclic groups includecholesterol and other steroid groups. Cationic lipids can be preparedwith a variety of counterions (anions) including among others: chloride,bromide, iodide, fluoride, acetate, trifluoroacetate, sulfate, nitrite,and nitrate.

[0075] A well-known cationic lipid isN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

[0076] DOTMA and the analogous diester DOTAP (1,2-bis(oleoyloxy)-3′(trimethylammonium) propane), are commercially available. Additionalcationic lipids structurally related to DOTMA are described in U.S. Pat.No. 4,897,355, which is herein incorporated by reference.

[0077] Another useful group of cationic lipids related to DOTMA andDOTAP are commonly called DORI-ethers or DORI-esters. DORI lipids differfrom DOTMA and DOTAP in that one of the methyl groups of thetrimethylammonium group is replaced with a hydroxyethyl group. Theoleoyl groups of DORI lipids can be replaced with other alkyl or alkenegroups, such as palmitoyl or stearoyl groups. The hydroxyl group of theDORI-type lipids can be used as a site for further functionalization,for example for esterification to amines, like carboxyspermine.

[0078] Additional cationic lipids which can be employed in the deliveryvectors or complexes of this invention include those described inWO91/15501as useful for the transfection of cells.

[0079] Cationic sterol derivatives, like3β[N-(N′,N′-dimethylaminoethane)carbamoyl] cholesterol (DC-Chol) inwhich cholesterol is linked to a trialkyammonium group, can also beemployed in the present invention. DC-Chol is reported to provide moreefficient transfection and lower toxicity than DOTMA-containingliposomes for some cell lines. DC-Chol polyamine variants such as thosedescribed in WO97/45442 may also be used.

[0080] Polycationic lipids containing carboxyspermine are also useful inthe delivery vectors or complexes of this invention. EP-A-304111describes carboxyspermine containing cationic lipids including5-carboxyspermylglycine dioctadecyl-amide (DOGS) anddipalmitoylphosphatidylethanolamine 5-carboxyspermylamide (DPPES).Additional cationic lipids can be obtained by replacing the octadecyland palmitoyl groups of DOGS and DPPES, respectively, with other alkylor alkene groups.

[0081] In the delivery vectors or complexes of the invention cationiclipids can optionally be combined with non-cationic co-lipids,preferably neutral lipids, to form liposomes or lipid aggregates.Neutral lipids useful in this invention include, among many others:lecithins; phosphatidylethanolamines, such as DOPE (dioleoylphosphatidylethanolamine), POPE(palmitoyloleoylphosphatidylethanolamine) and DSPE(distearoylphosphatidylethanol amine); phosphatidylcholine;phosphatidylcholines, such as DOPC (dioleoyl phosphatidylcholine), DPPC(dipalmitoylphosphatidylcholine) POPC (palmitoyloleoylphosphatidylcholine) and DSPC (distearoylphosphatidylcholine);phosphatidylglycerol; phospha-tidylglycerols, such as DOPG(dioleoylphosphatidylglycerol), DPPG (dipalmitoylphosphatidylglycerol),and DSPG (distearoylphosphatidylglycerol); phosphatidylserines, such asdioleoyl- or dipalmitoylphospatidylserine; diphospha tidylglycerols;fatty acid esters; glycerol esters; sphingolipids; cardolipin;cerebrosides; and ceramides; and mixtures thereof. Neutral lipids alsoinclude cholesterol and other 3DOH-sterols.

[0082] Moreover in the delivery vector or complexes of the invention oneor more amphiphilic compounds can optionally be incorporated in order tomodify its surface property. Amphiphilic compounds useful in thisinvention include, among many others; neoglycolipids such as GLU4 andGLU7 shown in FIG. 22, polyethyleneglycol lipids such asN-(ω-methoxy(polyoxyethylene)oxycarbonyl)-phosphatidylethanolamine,N-monomethoxy(polyoxyethylene)succinylphosphatidylethanol-amine andpolyoxyethylene cholesteryl ether; nonionic detergents such as alkylglycosides, alkyl methyl glucamides, sucrose esters, alkyl polyglycerolethers, alkyl polyoxyethylene ethers and alkyl sorbitan oxyethyleneethers and steroidal oxyethylene ethers; block copolymers such aspolyoxyethylene polyoxypropylene block copolymers.

[0083] In one aspect the cationic lipid of the present invention ismodified with a sugar moiety or a polyethylene glycol (PEG) moiety. In afurther aspect the complex of the invention further comprises a compoundcapable of acting as a cationic lipid, the compound comprising acholesterol group having linked thereto via an amine group, a sugarmoiety or a polyethylene glycol moiety. As demonstrated in the Exampleswe have found such sugar/PEG modified cationic lipids to be particularlyadvantageous. Thus in a further aspect the present invention provides acompound capable of acting as a cationic lipid, the compound comprisinga cholesterol group having linked thereto via an amine group, a sugarmoiety or a polyethylene glycol moiety. Preferably the compoundcomprises from 1 to 7 sugar moieties or a polyethylene glycol moieties.The compound may comprise a mixture of sugar moieties and polyethyleneglycol moieties. Preferably the sugar moiety is or is derived fromglucose or D-glucose.

[0084] D. Cationic Lipid/NOI/Packaging Polypeptide Complexes

[0085] A delivery vector/complex of the present invention is typicallymade by firstly contacting a packaging polypeptide and an NOI in asterile tube for about 10 mins at room temperature, resulting in acondensed polypeptide/NOI complex. A common technique is to spot thenucleic acid and protein alongside each other in the tube, but not incontact, and initiate mixing by adding a few hundred microlitres of aliquid carrier, such as a pharmaceutically acceptable carrier, excipientor diluent.

[0086] A further and preferred method of preparing a deliveryvector/complex of the present invention is by contacting a packagingpolypeptide and an NOI during continuous vortexing.

[0087] Typically a ratio of NOI to polypeptide at least 1:1, preferablyfrom 1:1 to 2:1, more preferably from 1.4:1 to 1.9:1, more preferablyfrom 1.5:1 to 1.8:1, is used. We have found a ratio of NOI topolypeptide of approximately 1:0.6 (˜1.7:1) to be particularlyeffective. In some aspects, typically a ratio of polypeptide to NOI offrom 0.2 to 1.5, preferably from 0.3 to 1.2 (w/w), more preferably from0.5 to 0.7 is used. In other embodiments the typically ratio ofpolypeptide to NOI is at least 10:1, or at least 20:1 (w/w). However,the optimum ratio may depend on the charge:amino acid ratio of thepackaging polypeptide. Generally, the lower the charge:amino acid ratio,the higher the polypeptide:NOI ratio used.

[0088] Next, cationic lipids are added to the complex. The cationiclipids may, in one embodiment, be part of a pre-formed liposomecomprising two or more lipid constituents, such as DC-Chol and DOPE. Thecationic lipids are typically incubated with the polypeptide/NOI complexfor about 20 mins at room temperature. A further and preferred method ofadding the cationic lipids is in the form of a cationic liposomesuspension. This final complex may be stored at approximately −80° C.with the addition of 10% sucrose (w/v) until use.

[0089] The amount of liposome to NOI is typically in the order of from3:1 to 20:1, preferably from 6:1 to 15:1, more preferably from 8:1 to14:1. We have found a ratio of liposome to NOI of 12:1 to beparticularly effective. In other embodiments the amount of liposome toNOI is typically in the order of the 2:1 to 10:1, or from 3:1 to 6:1.Where cationic lipids are used with neutral lipids, the ratio istypically in the order of 1:1.

[0090] In a highly preferred embodiment the ratioliposome:NOI:polypeptide is 3-20:1:0.5-1 preferably 8-14:1:0.5-0.7 morepreferably ˜12:1:˜0.6

[0091] The delivery vector/complex is now ready for use. Although it ispreferred to mix the various components in the order described above, itis possible to combine the components in any order. Where furtherpolypeptide components are to be added, they may be added at any stagebut preferably together with the packaging polypeptide.

[0092] It may be desirable to include other components within thevectors/complexes, for example ligands that bind to cell surfacereceptors, to provide the vectors/complexes with a degree of selectivityfor cell type. Ligands include peptides, glycoproteins,oligosaccharides, lectins and antibodies and fragments thereof.

[0093] E. Administration

[0094] The delivery vector/complex of the invention is preferablycombined with a pharmaceutically acceptable carrier or diluent toproduce a pharmaceutical composition (which may be for human or animaluse). Suitable carriers and diluents include isotonic saline solutions,for example phosphate-buffered saline. The composition of the inventionmay be administered by direct injection. The composition may beformulated for parenteral, intramuscular, intravenous, subcutaneous,intraocular or transdermal administration or inhalation. Typically, eachNOI may be administered at a dose of from 10 ng to 10 μg/kg body weight,preferably from 0.1 to 10 μg/kg, more preferably from 0.1 to 1 μg/kgbody weight.

[0095] Alternatively, transfection of patient cells may be carried outex vivo by removal of patient tissue, transfection using a deliveryvector/complex of the invention, followed by reimplantation of thetransfected tissue.

[0096] The routes of administration and dosages described are intendedonly as a guide since a skilled practitioner will be able to determinereadily the optimum route of administration and dosage for anyparticular patient and condition.

[0097] F. Uses

[0098] The delivery vectors/complexes in the present invention may beused to efficiently transfect eukaryotic cells, in particular mammaliancells, with NOIs. The delivery vectors/complexes have been shown to beparticularly efficient compared with prior art compositions intransfecting neuronal cells. This has specific implications for (i)research where neuronal cells are used and (ii) clinical applicationswhere it is desired to introduce NOIs into cells of the central ofperipheral nervous system of a human or animal. More generally, thedelivery vectors/complexes in the present invention may be used in avariety of NOI delivery applications such as gene therapy, DNA vaccinedelivery and in vitro transfection studies.

[0099] Examples of diseases that may be targeted for treatment using thecomplexes/vectors of the invention include diseases of the peripheral orcentral nervous system such as neurodegenerative diseases and damage tonervous tissue as a result of injury/trauma (including strokes). Inparticular, neurodegenerative diseases include motor neurone disease,several inherited diseases, such as familial dysautonomia and infantilespinal muscular atrophy, and late onset neurodegenerative diseases suchas Parkinson's and Alzheimer's diseases.

[0100] The delivery vectors/complexes of the invention may also be usedto administer therapeutic genes to a patient suffering from amalignancy. Examples of malignancies that may be targeted for treatmentinclude cancer of the breast, cervix, colon, rectum, endometrium,kidney, lung, ovary, pancreas, prostate gland, skin, stomach, bladder,CNS, oesophagus, head-or-neck, liver, testis, thymus or thyroid.Malignancies of blood cells, bone marrow cells, B-lymphocytes,T-lymphocytes, lymphocytic progenitors or myeloid cell progenitors mayalso be targeted for treatment.

[0101] The tumour may be a solid tumour or a non-solid tumour and may bea primary tumour or a disseminated metastatic (secondary) tumour.Non-solid tumours include myeloma; leukaemia (acute or chronic,lymphocytic or myelocytic) such as acute myeloblastic, acutepromyelocytic, acute myelomonocytic, acute monocytic, erythroleukaemia;and lymphomas such as Hodgkin's, non-Hodgkin's and Burkitt's. Solidtumours include carcinoma, colon carcinoma, small cell lung carcinoma,non-small cell lung carcinoma, adenocarcinoma, melanoma, basal orsquamous cell carcinoma, mesothelioma, adenocarcinoma, neuroblastoma,glioma, astrocytoma, medulloblastoma, retinoblastoma, sarcoma,osteosarcoma, rhabdomyosarcoma, fibrosarcoma, osteogenic sarcoma,hepatoma, and seminoma.

[0102] Other diseases of interest include diseases caused by mutations,inherited or somatic, in normal cellular genes, such as cystic fibrosis,thalessemias and the like.

[0103] Further areas of interest include the treatment of immune-relateddisorders such as organ transplant rejection and autoimmune diseases.The spectrum of autoimmune disorders ranges from organ specific diseases(such as thyroiditis, insulitis, multiple sclerosis, iridocyclitis,uveitis, orchitis, hepatitis, Addison's disease, myasthenia gravis) tosystemic illnesses such as rheumatoid arthritis, and other rheumaticdisorders, or lupus erythematosus. Other disorders include immunehyperreactivity, such as allergic reactions, in particular reactionassociated with histamine production, and asthma

[0104] The present invention will now be illustrated by means of thefollowing examples which are illustrative only and not limiting.

DESCRIPTION OF THE FIGURES

[0105]FIG. 1 shows a plate;

[0106]FIG. 2 shows a graph;

[0107]FIG. 3 shows a plate;

[0108]FIG. 4 shows a graph;

[0109]FIG. 5 shows a graph;

[0110]FIG. 6 shows a graph;

[0111]FIG. 7 shows a graph;

[0112]FIG. 8 shows a graph;

[0113]FIG. 9 shows structures;

[0114]FIG. 10 shows a graph;

[0115]FIG. 11 shows a graph;

[0116]FIG. 12 shows a graph;

[0117]FIG. 13 shows a graph;

[0118]FIG. 14 shows a graph;

[0119]FIG. 15 shows a graph;

[0120]FIG. 16 shows a plate;

[0121]FIG. 17 shows a graph;

[0122]FIG. 18 shows a graph;

[0123]FIG. 19 shows a structure;

[0124]FIG. 20 shows a reaction scheme;

[0125]FIG. 21 shows a reaction scheme;

[0126]FIG. 22 shows structures;

[0127]FIG. 23 shows principle of miscellar incorporation;

[0128]FIG. 24 shows a graph; and

[0129]FIG. 25 shows a graph.

DETAILED DESCRIPTION OF THE FIGS. 1 to 6

[0130]FIG. 1—The Adenoviral Core Protein Mu1 is More Efficient atBinding Plasmid DNA than Polyomavirus Core Protein Vp1

[0131] A) BSA has no effect on the electrophoretic mobility of pDNA. Onemicrogram of pCMVβ was incubated with 0 μg (lane 2), 5 μg (lane 3), 10μg (lane 4), 15 μg (lane 5), 20 μg (lane 6), 25 μg (lane 7) and 30 μg(lane 8) of BSA for 10 minutes at room temperature in 1× HBS. Sampleswere then analyzed on a 1% agarose gel for altered mobility. No changein electrophoretic mobility by BSA was detected.

[0132] B) In contrast to BSA, Mu1 peptide dramatically interfered withthe mobility of pDNA. pCMVβ (1 μg) was incubated with 0.25 μg (lane 2),0.5 μg (lane 3), 1 μg (lane 4), 2 μg (lane 4), 4 μg (lane 6), 6 μg (lane7) and 0 μg (lane 8) recombinant Mu1 peptide as in A. While ratios ofprotein to pDNA of 0.25 (w/w) (lane 2) did not alter migration of therelaxed form of pCMVβ (upper band) a slight retardation of supercoiledpDNA was seen (lower band). When ratios of 0.5 (w/w) or greater wereused, however, migration of both forms of pDNA was severely retarded.

[0133] C). The Polyomavirus protein Vp1 was much less efficient atpreventing pDNA migration. pCMVβ (1 μg) was incubated with 2 μg (lane2), 4 μg (lane 3), 6 μg (lane 4), 8 μg (lane 5), 16 μg (lane 6), 32 μg(lane 7) and 0 μg (lane 8) Vp1. Only ratios of 6 or higher (protein:pDNA, w/w) caused significant retardation of supercoiled pDNA (lane 6,lower band). Also, not until a ratio of 32 (w/w) was used was there anyeffect on relaxed pDNA (lane 7, upper band). In all gels lane 1corresponds to 1 Kb DNA marker (BRL).

[0134]FIG. 2—β Galactosidase Activity in ND7 Cells Transfected withpDNA-Mu1-Cationic Liposome Complexes

[0135] ND7 cells were seeded at a density of 5 ×10⁴ cells/well in 24well culture dishes 24 hrs prior to transfection. Immediately prior totransfection, cells were washed in serum-free media. Complexes wereformed by incubating pCMVβ with Mu1 prior to the addition of thecationic liposome DC-Chol/DOPE. In each case 1 μg pCMVβ was complexedwith 0.6, 6, 12, and 21 μg Mu1 peptide. Each of these combinations wasthen complexed with 3, 4 and 6 μg DC-Chol/DOPE. ND7 cells were exposedto transfection complexes for 2 hours then maintained at 37° C., 5% CO₂for another 24 hrs before being harvested and processed forβ-galactosidase enzyme assay. Numbers represent means±SD, n=3.

[0136]FIG. 3—Mu1 Enhances Cationic Liposome Mediated TransfectionEfficiency in the Neuronal Cell Line ND7

[0137] ND7 neurons were plated in 24 well culture dishes at a density of4×10⁴ cells/well and allowed to grow for 24 hrs. The undifferentiatedND7 neurons were then transfected with either pCMVb alone (A), pCMVbcomplexed with DC-Chol/DOPE (1/3, w/w) (B) or with pCMVb complexed withMu1 and DC-Chol/DOPE (1/12/6) (C). Forty-eight hours later the cellswere fixed and processed for histochemical detection of X-Gal. As can beseen in panel C inclusion of Mu1 in the complex at an optimal ratiosignificantly enhanced the number of X-Gal positive cells (blue).

[0138]FIG. 4—Mu1 is More Efficient at Enhancing Cationic LiposomeMediated Transfections in ND7 Cells than Vp1

[0139] pCMVβ plasmid DNA was complexed to various amounts ofpolycationic peptide and then mixed with cationic liposome at a ratio of1:3 (pCMVβ:liposome; w/w). After being washed briefly in serum freemedia, ND7 cells were exposed to the liposome-polycation-liposomecomplexes for two hours and then returned to serum containing mediaTwenty-four hours later the cells were harvested and processed forβ-galactosidase enzyme assay. Each condition was performed in triplicateand each experiment replicated three times. Numbers represent means±SD.

[0140]FIG. 5—Mu1 Enhances DC-Chol/DOPE Transfection in COS-7 Cells

[0141] COS cells were seeded at a density of 60-80% confluence in 24well culture dishes 24 hrs prior to transfection. Immediately prior totransfection, cells were washed in serum-free media Incubating pCMVβwith Mu1 prior to the addition of the cationic liposome DC-Chol/DOPEformed complexes capable of cellular transfection. In each case 1 μgpCMVβ was complexed with 12 μg Mu1 peptide that had been found optimalfor ND7 cells. The pCMVβ:Mu1 complexes were then mixed with 3, 4 and 6μg DC-Chol/DOPE. COS cells were exposed to transfection complexes for 2hours then maintained at 37° C., 5% CO₂ for another 24 hrs before beingharvested and processed for β-galactosidase enzyme assay. Numbersrepresent means±SD, n=3.

[0142]FIG. 6—Transfection Efficiency in Differentiated ND7 Cells withpCMVβ-Mu1-Cationic Liposome Complexes

[0143] ND7 cells were plated in a 24 well culture plate at a density of4×10⁴ cells per well in normal growth media (+serum). Twenty-four hourslater the media was replaced with differentiation media and the cellswere grown for an additional 24 hrs. Three different differentiationmedias were used; serum-free (−serum), normal growth media plus 1 mMcAMP (cAMP), or reduced serum (0.5%) plus 1 mM cAMP and 50 ng/ml nervegrowth factor (NGF). The cells were then transfected with pCMVbcomplexed with either DC-Chol/DOPE alone or Mu1 plus DC-Chol/DOPE.Forty-eight hours later the cells were fixed and processed for X-Galhistochemistry and the percentage of positive cells determined. In allcases the. presence of Mu1 increased the number of positive cells.

[0144] Interestingly the number of cells transfected was greater bothwith and without Mu1 for cells grown in cAMP.

EXAMPLES

[0145] Materials and Methods

[0146] Peptide Synthesis

[0147] Peptides Vp1 and Mu1 were synthesized on a Shimadzu PSSM-8 solidphase peptide synthesizer using a five-fold excess of(9-fluorenyl)methoxycarbonyl (Fmoc)-protected L-amino acids(Novabiochem) and the FastMoc™ reagents2-(1H-benzotriazole-1-yl)-1,1,3,3-tetra-methyluroniumhexafluorophosphate/hydroxybenzotriazole (HBTU/HOBt) (Advanced ChemtechEurope) as the amide coupling agent. After resin cleavage anddeprotection, desalting was performed by gel filtration using a columnof P2 Biogel (2×28 cm; Biorad) attached to an FPLC system (AmershamPharmacia Biotech UK) with 0.1% aqueous TFA as eluant at a flow rate of0.5-0.75 ml/min. Final preparative reverse-phase purification wasachieved with a Vydac column (C18, 5 μm, 2×25 cm; Hichrom) attached to aGilson HPLC system (Anachem). Peptides were eluted at 5ml/min by meansof a linear gradient of acetonitrile in 0.1% aqueous TFA and elutionmonitored at 220-230 nm.

[0148] The Vp1 peptide was prepared using a preloadedL-Pro-2-chlorotrityl super acid labile resin (Novabiochem) (100 mg, 1.05mmol/g, 0.1 mmol). Extended coupling times were used to incorporate allamino acid residues from the sixth (Lys) through to the N-terminalresidue. After automated N-terminal Fmoc deprotection with piperidine(20%, v/v) in dimethyl formamide, the resin was isolated, washed withdimethylformamide (10 ml) and methanol (15 ml), and then dried in vacuo.Crude peptide was cleaved from the resin using ice cooled TFA (8 ml),containing phenol (7%, w/v), ethanedithiol (2%, v/v), thioanisole (4%,v/v) and water (4%, v/v) (known as Mixture A), and then precipitatedwith ice cold methyl-tert-butylether (MTBE) (30 ml). The subsequentpellet was then desalted and the crude peptide mixture purified byreverse phase HPLC. After elution, fractions containing the desiredpeptide (eluting with acetonitrile 68.5% v/v) were combined andlyophilized to give the peptide as a white powder. Overall yield: 32 mg(15 μmol, 15%); MS (MALDI-TOF) C₈₅H₁₅₁N₂₆O₂₆S₃: [M+H]⁺ calcd 2049.5,found 2050.2. The sequence was confirmed by amino acid composition andsequence analysis. Homogeneity was judged >95% by HPLC analysis.

[0149] The Mu1 peptide was prepared using Gly-Wang resin (Novabiochem)(40 mg, 0.67 mmol/g, 0.03 mmol). Normal coupling times were usedthroughout After automated N-terminal Fmoc deprotection as above, theresin was isolated and washed with dichloromethane (20 ml) and methanol(20 ml) after which the resin was dried in vacuo. Crude peptide wascleaved from the resin using Mixture A (8 ml) and precipitated with MTBE(30 ml), all as above. Finally, the crude peptide mixture was desaltedand purified by reverse phase HPLC. After elution, fractions containingthe desired peptide (eluting with acetonitrile 17.2%) were combined andlyophilized to give the peptide as a white powder. Overall yield: 65 mg(26 μmol, 80%); MS (ES) C₉₅H₁₇₀N₅₂O₂₁S₂: [M+H]⁺ calcd 2440.7, found2440.6. Homogeneity was judged >95% by HPLC analysis.

[0150] DNA Binding Analysis

[0151] The purified peptides were reconstituted in sterile distilled H₂Oat 3 mg/mL. Peptide and pDNA were complexed in 20 μL HEPES bufferedsaline (137 mM NaCl, 5 mM KCl, 0.75 mM Na₂HPO₄, 19 mM HEPES, pH 7.4) for20 minutes at room temperature. Peptide: pDNA complexes weresubsequently analyzed by agarose gel electrophoresis (1%). Controlincubations for general macromolecular pDNA interactions were performedwith varying amounts of molecular biology grade purified bovine serumalbumin (Sigma).

[0152] Cell Cultures

[0153] ND7s are a well-characterized cell line derived from the fusionof a neuroblastoma N18Tg2) with neonatal rat sensory neurons ²⁸. Thecell line was maintained in normal growth media (NGM) (Leibovitz's L-15media (BRL) enriched with 10% Fetal bovine serum (BRL), 4 g/L glucose, 4g/L sodium bicarbonate (BRL), 100 IU/mL penicillin/streptomycin (BRL))at 37° C. and 5% CO₂. The cells were plated onto 24 well plates (Costar)at a density that produced 70% confluence after 24 hours.

[0154] Differentiation of ND7 cells was carried out using threepreviously described methods ^(28, 29). ND7 cells were seeded in NGM ata density of 4×10⁴ cells per well in a 24 well culture dish (Nunc).Twenty four hours later the media was replaced with either: a) NGMsupplemented with 1 mM adenosine 3′:5′-cyclic monophosphate (cAMP;Sigma), or b) serum-free differentiation media (50% Hams F12, 50% DMEM,5 μg/mL Transterrin, 250 ng/mL Insulin, 0.3 μM sodium selenite), or c)low serum nerve growth factor (NGF) media (L-15 supplemented with 2 mMglutamine, 4 g/L glucose, 4 g/L sodium bicarbonate, 10 u/mL penicillin,10 g/mL streptomycin, 0.5% FCS, 1 mM cAMP, 50 ng/mL NGF (Alomone Labs)).Differentiated ND7 cells were grown in appropriate media for 24 hrs at37° C., 5% CO₂ prior to transfection.

[0155] COS-7 cells (derived from Green Monkey kidney) were grown in RPMI1640 media (BRL) supplemented with 10% fetal bovine serum (BRL) and 100IU/mL penicillin/streptomycin (BRL).

[0156] Plasmid Constructs

[0157] All transfections utilized the reporter plasmid pCMVβ(Clontech,Palo Alto, Calif.) containing the full-strength sequence for E. coliβ-galactosidase downstream of the human cytomegalovirus immediate-earlypromoter/enhancer (Clontech). Stocks of plasmid DNA were prepared usingstandard molecular cloning techniques and purified using the QiagenEndotoxin-free plasmid purification system (Qiagen, Dorking, UK).

[0158] Liposomes

[0159] DC-Chol/DOPE liposomes were prepared as previously described^(30, 31). Briefly, 6 μmol of DC-Chol and 4 μmol of DOPE (supplied at 10mg/mL in CHCl₃) were added to freshly distilled CH₂Cl₂ (5 mL) undernitrogen. 5 mL of 20 mM Hepes (pH 7.8) was added to the mixture and thiswas sonicated for 3 minutes. The organic solvents were removed underreduced pressure and the resulting liposome suspension was thensonicated for a further 3 minutes. Liposome preparations were stored at4° C.

[0160] Transfection Protocol

[0161] Since initial experiments determined that the presence of fetalbovine serum inhibited transfection of ND7 cells serum-freedifferentiation media was used for all transfections. Various amounts ofDNA and liposome were placed in the bottom of a 7 mL sterile Bijoucontainer (Bibby Sterilin Ltd., Staffordshire, U.K.), but not in contactwith each other.

[0162] DNA and liposomes were combined by the addition of 400 μLserum-free differentiation media and gentle shaking. The DNA: liposomemixture was incubated at room temperature for 20 to 30 minutes beforebeing applied to the cells. The DNA/liposome mixture was then applied tothe cells and incubated at 37° C., 5% CO₂ for 2 hours after which thismedia was replaced with complete media Twenty four to 48 hours later thecells were fixed and processed for X-gal histochemistry as described ³¹or harvested for β-galactosidase enzyme assays (Promega Corp.).

[0163] Cell counts were performed under ×40 magnification using a NikonDiaphot inverted microscope. Each transfection was repeated at leastthree times and at least three separate counts were made for each well.

[0164] Transfection complexes including the test peptides were generatedin the following manner. Various amounts of peptide was placed in thebottom of sterile polystyrene containers alongside, but not in contactwith 1 μg·pCMVβ and mixed by adding 400 μl serum free NGM media Thecomplexes were incubated at room temperature for 10 minutes after whichDC-Chol/DOPE was added. The pDNA/peptide/liposome complex was furtherincubated at room temperature for 20 minutes and then administered tocells as above.

Example 1 DNA Binding Analysis

[0165] Mu1 is a polycationic peptide comprised of 19 amino acidsassociated with the core complex of Adenovirus (Table 1) ^(27, 32). Wecompared the DNA binding capacity of Mu1 with the mouse polyomavirusmajor capsid protein Vp1 by interaction with plasmid DNA in a gelretardation assay. Vp1 is a 19 amino acid peptide that contains anuclear localization signal ²⁶ and contains fewer positively chargedamino acids than Mu1. It was therefore predicted to have a lower DNAbinding capacity. TABLE 1 Mu1 and VP1 protein sequences Charge/AAPolypeptide Sequence MW ratio Mu1NH₂—Met—Arg—Arg—Ala—His—His—Arg—Arg—Arg—Arg— 2440 0.63Ala—Ser—His—Arg—Arg—Met—Arg—Gly—Gly—OH VP1NH₂—Met—Ala—Pro—Lys—Arg—Lys—Ser—Gly—Val—Ser—Lys— 2049 0.26Cys—Glu—Thr—Lys—Cys—Thr—Pro—Pro—OH

[0166] The NLS sequence in VP1 is underlined

[0167] Varying amounts of purified peptide were incubated at roomtemperature in HBS for approximately 10 minutes and then analyzed byagarose gel electrophoresis. Without the addition of peptide,supercoiled and relaxed circular plasmid DNA (pDNA) migrated in theexpected manner (FIG. 1, lane 8).

[0168] Beginning at a DNA: Mu1 peptide ratio of 1:0.25 (w/w) themigration of plasmid DNA was retarded (FIG. 1). The migration of plasmidDNA was slightly affected at 1:0.25 ratios (w/w), but at a ratio 1:0.5(w/w) the migration of plasmid DNA was severely slowed and very littlemanaged to migrate out of the wells. At ratios of 2:1 and above pDNA wasunable to migrate into the agarose gel and the ability of ethidiumbromide to interchelate into the plasmid was reduced. In contrast toMu1, no effect on the electrophoretic mobility of plasmid DNA wasdetected with Vp1 at pDNA: protein ratios up to 1:8 (w/w) (FIG. 1). Theaddition of 8 μg Vp1 to 1 μg plasmid DNA resulted in a broadening of thesupercoiled pDNA band. However, no effect was seen on the relaxed pDNAband with Vp1 until a ratio of 1:32 (w/w) pDNA: protein was used. Atthis ratio, both supercoiled and relaxed pDNA bands were significantlyretarded and some DNA could be seen retained in the well. No effect onelectrophoretic mobility was detected when pDNA was incubated with BSAat ratios up to 1:30 (w/w) pDNA: protein (FIG. 1).

Example 2 Transfection in Undifferentiated ND7s

[0169] We examined the ability of Mu1 and Vp1 to enhance thetransfection of a neuronal cell line by cationic liposomes using aβ-Galactosidase reporter gene assay. ND7 cells were transfected withpCMVβ complexed to varying amounts of peptide and DC-Chol/DOPE. We havepreviously shown that the cationic liposome DC-Chol/DOPE is capable ofefficiently transfecting the neuronally derived ND7 cell line ³¹. Inthis study we found that optimal efficiencies (>40%) were obtained inthis neuronally derived cell line using 1 μg plasmid DNA complexed with3 μg DC-Chol/DOPE ³¹. Temporally, maximal levels of transgene expressionare obtained between 48-60 hours post transfection. Therefore, in orderto maximize the chance of detecting improvements in transfection weperformed all our assays within 12-20 hours of transfection at a timewhen levels of reporter gene expression were lower. Previously we founda pDNA: liposome ratio of 1:3 (w/w) optimal for transfections in ND7cells ³¹. We therefore compared the effect of various amounts of peptideon transfections at ratios of 1:3, 1:4 and 1:6 pDNA: DC-Chol/DOPE. Thegel retardation analysis suggested that an approximate ratio of 1:0.5(w/w), pDNA: Mu1, was enough to essentially bind all of the plasmid DNA(FIG. 1). However, initial experiments using this ratio and liposomesdid not affect transfection efficiencies (not shown). The volumes usedto generate transfection complexes were much larger than those used toperform the gel retardation assay (400 μL vs. 20 μL). Therefore wetested larger quantities of Mu1 that were of a similar concentration insolution as that used in the gel retardation assay. We compared theeffect 0.6, 6, 12 and 21 μg of Mu1 peptide would have on DC-Chol/DOPEmediated transfections. We found that Mu1 was able to improve cationicliposome mediated transfection efficiencies over 4-fold. The greatestimprovement in transfection efficiencies occurred when the relativeratios of 1/12/6, pCMVβ/Mu1/(DC-Chol/DOPE) (w/w/w) were used. Thiscombination led to an 11-fold increase in transfections compared to DNAalone (FIG. 2).

[0170] The β-galactosidase reporter gene assay provides a measure of theoverall level of β-galactosidase produced, but gives no informationregarding the number of cells transfected. For this reason, we alsoperformed cell counts on transfected ND7 cells. Cells were seeded at adensity of 4×10⁴ in 24 well culture plates. After 24 hours the cellswere washed briefly in serum-free media and transfected with pCMVβcomplexed to DC-Chol/DOPE and Mu1 peptide. The ratios used were thosefound to be optimal in the reporter gene assay, 1:12:6,pCMVβ:Mu1:DC-Chol/DOPE. Using these ratios, we found a 6-fold increasein the number of β galactosidase positive cells (FIGS. 3 & 6). Noobvious cell loss was detected with the Mu1 complex at any of theconcentrations utilized. Similarly the concentration of protein in thecellular lysates used for β-Galactosidase reporter gene assay did notsignificantly vary with untransfected cells (data not shown).

[0171] In contrast, no improvement on transfection efficiency could befound with Vp1 (FIG. 4). No improvement in transfection efficienciesover naked DNA was seen with pCMVβ complexed to Mu1 alone.

[0172] In order to see whether improved transfections could be achievedin other cell types we performed a similar analysis on COS-7 cells. Mu1also improved liposome-mediated transfection in COS-7 cells (FIG. 5).The same ratio of pDNA:Mu1:liposome optimal for ND7 cells was best forCOS-7, cells. A similar degree of improvement was also seen (3.7 fold)over cationic liposomes alone.

Example 3 Transfection in Differentiated ND7s

[0173] We also examined the ability of Mu1 to improved cationicliposome-mediated transfection in differentiated ND7s. The ND7 cell lineis derived from a fusion of primary rat dorsal root ganglia (DRG)neurons and the mouse neuroblastoma N18Tg2 ²⁸. ND7 cells can bedifferentiated in a variety of manners including the withdrawal ofserum, cAMP administration or exposure to reduced serum plus cAMP andnerve growth factor. Differentiation of ND7s leads to the expression ofcellular properties associated with their parental nociceptive sensoryneurons including a reduction in cell division and the onset of neuriteoutgrowth. ND7 cells were seeded in 24 well culture plates and 24 hourslater differentiated. Fifteen to 20 hours following the onset ofdifferentiation, they were transfected as above. Fifteen to 20 hoursfollowing transfection, cells were fixed and processed for X-galhistochemistry. Consistent with previous observations, transfectionefficiencies varied greatly between the three differentiated groups. ND7cells differentiated by withdrawal of serum exhibited the lowest levelsof transfection (1.3%) while highest levels were seen in the cAMP group(8%) and intermediate levels in the low serum/cAMP/NGF group (4.7%)(FIG. 5). In all three conditions, however, inclusion of Mu1 polypeptidein the transfection complex improved the transduction of differentiatedND7 cells. ND7s differentiated by either cAMP alone or exposure to lowserum/cAMP/NGF exhibited increased efficiencies of greater than 6 fold(FIG. 5). The greatest improvement in efficiencies was seen, however, inthe group differentiated by serum withdrawal. Here, increases of greaterthan 10 fold were observed.

[0174] Complexes of Mu1 Peptide and DNA

[0175] As shown in Example 1 (DNA binding Analysis) using gelelectrophoresis, the migration of plasmid DNA was severely retarded andlittle DNA migrated out of the wells above a Mu1:DNA 0.5:1.0 (w/w). Thisimplied that Mu1 peptide was strongly interacting with DNA and mightneutralise and condense nucleic acids to form small particles suitablefor gene delivery. The size of Mu1:DNA (MD) particle sizes were examinedover the Mu1:DNA ratio range indicated in FIG. 7.

[0176] MD particles were prepared by mixing. Briefly, appropriatealiquots of Mu1 peptide in deionized water were added to plasmid DNA(pCMVβ) (final concentration 220μg/ml) in 20 mM Hepes buffer, pH7.0.After mixing well, each mixture was incubated for 10 min at 20° C.Immediately after incubation each mixture was diluted with the Hepesbuffer (final DNA concentration 24 μg/ml) and subjected to particle sizeanalysis by photon correlation spectroscopy (N4 plus, Coulter). Allmeasurements were performed at 20° C. and data collected at an angle of90°. Unimordal analysis was used to calculate the mean particle size andstandard distribution (S.D.).

[0177] Interestingly, though Mu1 bound DNA and formed complexes over thecomplete range examined, the particle size varied considerably inresponse to the Mu1:DNA ratio. Stable, small nano-particles were formedwithin the Mu1:DNA ratio 0.3 to 1.2 ( range L) and over 5 (range H).Intermediate ratios resulted in heavy aggregation with the size ofcomplex particles growing over the time of incubation to reach more than2 μm in size (FIG. 7).

Example 4 Preparation of LMD in the Range L

[0178] We determined whether liposome-Mu1-DNA complexes with a lowMD:DNA ratio could form stable nano-particles and whether the resultingcomplex particles could have good transfection activities.

[0179] Preparation of liposomes; DC-Chol (30 μmol) and DOPE (20 μmol)were combined in dichloromethane. The organic solvent was removed underreduced pressure using a rotary evaporator and the residue dried for 3 hin vacuo. Following this, 4 mM Hepes buffer, pH7.0 (3 ml), was added tothe lipid film with vortex mixing. After brief sonication (2-3 min), theresulting cationic liposome suspension was extruded by means of anExtruder device (Lipex Biomembranes) three times through two stackedpolycarbonate filters (0.2 μm Millipore) and then ten times through twostacked polycarbonate filters (0.1 μm Millipore) to form small liposomes(109 nm average diameter by PCS) (approx. 8-10 mg/ml depending upon thepreparation).

[0180] Preparation of Liposome:Mul:DNA (LMD) complexes; Mu1 peptide(0.12 mg in deionised water, peptide concentration 3.5 mg/ml) was addedto a solution of plasmid DNA (pCF1-CAT) (0.2 mg, plasmid concentrationtypically 1.0 mg/ml) in 4 mM Hepes buffer during continuous vortexing.Cationic liposome suspension (total lipid 2.4 mg, 4 μmol) was thenintroduced resulting in the formation of small particles with narrowsize distribution (168 nm±58 nm) as measured by PCS. This LMD (final DNAconcentration 0.14 mg/ml) was stored in −80° C. with the addition of 10%sucrose (w/v) until use. No particle size deviation was observed overone month.

[0181] Liposome:DNA (LD) complexes (lipoplexes) were prepared forcontrol experiments with a Liposome:DNA ratio of 3:1 (w/w), the optimalcomposition for transfection of ND7 cells.

[0182] Transfection in ND7 cells; ND7 cells were seeded in normal growthmedium (NGM) (with 10% serum) at a density of approximately 4×10⁴ cellsper well, in a 24-well culture plate. After 24 h, cells were washed bybrief exposure to NGM (serum free) and then treated with solutionscontaining LMD or LD complexes, prediluted with NGM (serum free) (finalDNA concentration 3.2 μg/ml in all cases), for the time periodsindicated. Cells were then washed again and incubated for a further 48 hprior to harvesting. Levels of transfection were determined bychloramphenicol transferase (CAT) enzyme assay using ¹⁴C-CAM assubstrate (Promega). Transfection activity was expressed as a percentage(%) conversion of the imputed ¹⁴C-CAM by the enzyme.

[0183] We found much higher reporter gene expression with LMD comparedto LD mediated transfection. In fact, LMD transfection resulted in 16times more CAT enzyme activity after a transfection time of 10 mins, and6 times as much after a transfection time of 60 min compared withLD-mediated transfection (FIG. 8). Significant transfection was observedwith LMD even when the transfection time was as short as 10 min. Thisdata illustrates how rapidly LMD particles are able to enter cells.

Example 5 Cationic Lipid (Cytofectin) Variations

[0184] We determined whether LMD complexes could be bettered byincorporating poly cationic cholesterol lipids (WO 97/45442). CDAN(B198), ACHx (CJE52) and CTAP (B232) (FIG. 9) were used to make cationicliposomes in place of DC-Chol. Each cationic liposome system used wascomposed of 60 mol % of cationic lipid and 40 mol % of DOPE and preparedas described in Example 4. The following different LMD complex systemswere prepared and compared: LMD(DC-Chol), LMD(B198), LMD(CJE52), andLMD(B232). All LMD systems were prepared with cationic liposomes (totallipid 20 μmol) and 0.6 mg of Mu1 peptide per 1.0 mg of DNA (pCMVβ), asdescribed above. Particles were shown to be under 200 nm in diameter.

[0185] Liposome:DNA (LD) complex mixtures (lipoplexes) were prepared forcontrol experiments with a Liposome:DNA ratio of 3:1 (w/w), the optimalcomposition for transfection of ND7 cells.

[0186] Transfection in ND7 cells; ND7 cells were seeded in NGM (with 10%serum) at a density of approximately 4×10⁴ cells per well, in a 24-wellculture plate. After 24 h, cells were washed by brief exposure to NGM(serum free) and then treated with solutions containing LMD or LDcomplexes, prediluted with NGM (serum free) (final DNA concentration 2.5μg/ml in all cases), for 1 h. Cells were then washed again and incubatedfor a further 48 h prior to processing for histochemical staining withX-gal. The number of cells stained blue were counted under an invertedmicroscope.

[0187] In all cases LMD formulations worked better than corresponding LDsystems prepared with the same poly cationic cholesterol lipids (FIG.10). The rank order in transfection efficiency wasLMD(B198)>LMD(DC-Chol)>LMD(CJE52)>>LMD(B232). The same rank order,B198>DC-Chol>B232, was observed with corresponding LD systems.

Example 6 Amount of Mu1 Peptide in the Range L

[0188] We examined the effect of Mu1:DNA ratio (at the range L in FIG.7) on transfection activity.

[0189] Cationic liposomes composed of cationic lipid B198 and DOPE (3:2m/m) were prepared as the same manner described in Example 4. A seriesof MD complex mixtures (Mu1:DNA ratio varying from 0.3 to 1.2) wereprepared and complexed with the cationic liposome. The resulting LMDsystems were comprised of liposome:Mu1:DNA (pCMVβ) in ratios of12:0.3:1, 12:0.6:0.6, 12:0.9:1 and 12:1.2:1 w/w/w respectively. Measuredsizes of LMD particles were approximately 150 nm.

[0190] Transfection activities were evaluated in vitro using Panc-1cells (human pancreatic cancer cell line). The cells were seeded at anapproximate density of 5×10⁴ per well in a 24-well culture plate in RPMIsupplemented with 10% FCS and grown for 24 h in the presence of 5% CO₂at 37° C. Cells were washed by brief exposure to RPMI and then treatedwith solutions of LMD complexes, prediluted with RPMI (final DNAconcentration 5.0 μg/ml in all cases), for 30 min. Cells were thenwashed again and incubated for a further 48 h in RPMI supplemented with10% FCS prior to harvesting and the assay of β-galactosidase enzymeactivity using a standard assay kit (Promega).

[0191] As shown in FIG. 11, the optimum liposome:Mu1:DNA ratio fortransfection of Panc1 cells was found to be 12:0.6:0.6. Otherwise,excellent transfection results were obtained with these low ratio Mu1LMD complexes.

Example 7 Amount and Composition of Lipids

[0192] To investigate the effect of varying the ratio of cationic lipidto DOPE as well as the ratio of total lipid to Mu1 and DNA, a series ofLMD systems were prepared using B198 as the preferred cationic lipid.Cationic liposomes composed of 60 mol % of B198 and 40 mol % of DOPE(3:2 m/m), 50 mol % of B198 and 50 mol % of DOPE (1:1 m/m) and 33 mol %of B198 and 67 mol % of DOPE (1:2 m/m) were prepared and combined with astandard MD complex mixture (Mu1:DNA 0.6:1 w/w) at ratios indicated inFIG. 12, according to the method in Example 4.

[0193] Liposome:DNA (LD) complex mixtures (lipoplexes) were prepared forcontrol experiments with a Liposome:DNA ratio of 3:1 (w/w). All LMDsystems were found to have a larger average size when lower amounts ofcationic liposomes were complexed with MD complexes. However, the sizeof LMD particles composed of more than 12 μmol lipids/mg DNA remainedless than 200 nm, whilst that of 12 to 6 μmol lipids/mg DNA climbedabove that value. Occasionally, visible aggregation was observed duringthe preparation of LMD systems comprised of 6 μmol lipids/mg DNA.

[0194] Transfection activities were determined with Panc-1 cells (FIG.12). The cells were seeded at an approximate density of 5×10⁴ per wellin a 24-well culture plate in DMEM supplemented with 10% FCS and grownfor 24 h in the presence of 5% CO₂ at 37° C. Cells were washed by briefexposure to DMEM and then treated with solutions containing LMD or LDcomplexes, prediluted with DMEM (final DNA concentration 5.0 μg/ml inall cases), for 2 h. Cells were then washed again and incubated for afurther 48 h in DMEM supplemented with 10% FCS prior to harvesting andassay of β-galactosidase enzyme activity using a standard assay kit(Promega).

[0195] The maximum transfection activity was not significantly differentwith the three cationic different lipid to DOPE formulations tested. Inthe case of LMD systems prepared with B198:DOPE (1:2 m/m) the maximumtransfection was achieved at a liposome:DNA ratio of around 12.5 μmollipid /mg DNA. The transfection activities of LMD systems prepared withB198:DOPE (3:2 m/m) and B198:DOPE (2:2 m/m) cationic liposomes reached aplateau at liposome:DNA ratios greater than 12.5 μmol lipid/mg DNA. AllLMD systems analysed tended to show low transfection activities at lowliposome:DNA ratios (FIG. 12). It is considered that LMD formulationscomposed of low amounts of Mu1 peptide should need larger amounts ofcationic liposomes compared to the formulations prepared with higheramounts of Mu1 peptide in order for the respective LMD systems to showfull transfection activity.

Example 8 Comparison of Mu1 Peptide with Protamine

[0196] Protamine is a naturally occurring cationic peptide abundant inpiscine sperm and is potent in neutralising and condensing DNA. Thetransfection activity of protamine was compared with that of Mu1peptide. Mu1 peptide or protamine sulfate (Sigma, grade X from Salmon)was complexed with DNA (pCMVβ) and then cationic liposomes (B 198:DOPEin a ratio of 3:2 m/m) giving a liposome:peptide:DNA ratio of 12:0.6:1(w/w/w).

[0197] The transfection activities were examined in Swiss 3T3 cells. Thecells were seeded at an approximate density of 2×10⁴ per well in a24-well culture plate in DMEM supplemented with 10% FCS and grown for 48h to complete confluence in the presence of 5% CO₂ at 37° C. Cells werewashed by brief exposure to DMEM and then treated with solutionscontaining LMD or LD complexes, prediluted with DMEM (final DNAconcentration 5.0 μg/l in all cases), for 1 or 2 h. Cells were thenwashed again and incubated for a further 48 h in DMEM supplemented with10% FCS prior to harvesting. The level of β-galactosidase enzymeactivity was determined with a standard assay kit (Promega).

[0198] As shown in FIG. 13 the complexes comprising Mu1 peptide showedbetter transfection of these confluent cells than those comprisingprotamine.

Example 9 Alternative Cationic Peptides

[0199] In order to examine the effects of various alternative cationicpeptides on transfection activities, a series of liposome:cationicpeptide:DNA complexes were prepared and their relative transfectionabilities analysed in vitro. The peptides used were poly-lysinehydrochloride (average molecular weight 3970, Sigma), poly argininehydrochloride (average molecular weight 11800, Sigma) a peptide derivedfrom protein V, pV (p5, sequence shown below), a peptide analogue of Mu1(V, sequence shown below) and Mu1 peptide itself. The p5 peptide and Vpeptide were synthesized using the same solid-phase peptide synthesismethodology as was used to prepare Mu1 peptide.

[0200] Each peptide was combined with cationic liposome (DC-Chol:DOPE3:2 m/m) and DNA (pCMVβ) in the liposome:peptide:DNA ratio of 12:0.6:1(w/w/w) as described in Example 4. The transfection activities wereexamined using HeLa cells (human epithelial cells). The cells wereseeded at an approximate density of 5×10⁴ per well in a 24-well cultureplate in DMEM supplemented with 10% FCS and grown for 24 h in thepresence of 5% CO₂ at 37° C. Cells were washed by brief exposure to DMEMand then treated with solutions containing LMD or LD complexes,prediluted with OPTIMEM (Gibco) (final DNA concentration 1.0 μg/ml inall cases), for 30 min. Cells were then washed again and incubated for afurther 48 h in DMEM supplemented with 10% FCS prior to harvesting. Thelevel of β-galactosidase enzyme activity was determined with a standardassay kit (chemiluminescent, Roche).

[0201] As shown in FIG. 14, the cationic peptides derived fromadenovirus (Mu1 and p5) and the Mu1 analogue (V) revealed excellenttransfection activity compared to complexes prepared using the syntheticcationic polypeptides, poly lysine and poly arginine.

[0202] Amino Acid sequences of p5, V and Mu1 peptide P5;RPRRRATTRRRTTTGTRRRRRRR V; VRRVHHRRRRVSHRRVRGG Mu1; MRRAHHRRRRASHRRMRGG

Example 10 Comparison with Transfast using Panc-1

[0203] LMD and LD were prepared by the same method described in Example4 except for use of pCMVβ. Transfast (Promega) DNA complex was preparedaccording to manufacturer's protocol.

[0204] Transfection activities were evaluated in vitro using Panc-1cells. The cells were seeded at an approximate density of 5×10⁴ per wellin a 24-well culture plate in RPMI supplemented with 10% FCS and grownfor 24 h in the presence of 5% CO₂ at 37° C. Cells were washed by briefexposure to RPMI and then treated with solutions containing LMD or LDcomplexes, prediluted with RPMI (final DNA concentration 5.0 μg/ml inall cases), for the times indicated. Cells were then washed again andincubated for a further 48 h in RPMI supplemented with 10% FCS prior toharvesting. The level of β-galactosidase enzyme activity was determinedwith a standard assay kit (Promega). Transfection with Transfast:DNAcomplex was performed in serum free medium (optimum conditions) for 1 h.

[0205] As shown in FIG. 15, LMD showed better transfection activity thanthe Transfast:DNA complex and LD. These results are completelyconsistent with those found with ND-7 cells.

Example 11 Comparison with Lipofectamine using Human Bronchial Cells

[0206] The transfection activity of LMD complexes was compared with thatof Lipofectamine (Gibco) complexed with DNA using HBE cells (humanbronchial epithelium cell).

[0207] The cells were seeded in a 12-well culture plate in DMEMsupplemented with 10% FCS and grown for 24 h in the presence of 5% CO₂at 37° C. Cells were washed by brief exposure to DMEM and then treatedwith solutions containing either LMD (prepared as in Example 4) or LD(prepared from lipofectamine:DNA 12:1 w/w) complexes, prediluted withOPTIMEM (Gibco) (final DNA concentration 5.0 μg/ml in all cases), forthe indicated times (see FIG. 16). Cells were then washed again andincubated for a further 48 h in DMEM supplemented with 10% FCS prior toprocessing for histochemical staining with X-gal.

[0208] LMD showed a better transfection activity than lipofectamine(FIG. 16) and exhibited a more rapid uptake by HBE cells. Similarresults were seen with ND7 and Panc-1 cells.

Example 12 Comparison with LT1 using Rat Brain; Ex Vivo Experiment

[0209] We assessed transfection activities in organotypic cultures fromthe rat brain using a reporter DNA (pCMVβ) in order to mimic an in vivomodel. Brain slices were maintained on transparent porous membranes andwere observed to maintain their intrinsic connectivity andcytoarchitecture to a large degree.

[0210] LMD and LD were prepared as shown in Example 4. LT1 is apolyamine transfection reagent manufactured by PanVera Co. A complexcontaining cationic liposome (DC-Chol:DOPE, 3:2 m/m), LT-1 andpCMVβplasmid in the ratio 3:3.2:1 (w/w/w) was prepared. Brain sliceswere treated with solutions containing LMD, LD or liposome:LT1:DNA for 2h (Murray et al., Gene Ther. 1999, 6, 190-197). In all cases nomorphological changes in the sections were observed during theexperiment. After 48 h incubation post-transfection, cells wereharvested, X-gal stained and the number of blue cells counted on a slice(FIG. 17).

[0211] At a DNA dose of 5.0 μg (2 ml culture), LMD gave an apparentlylarger number of blue stained cells than LD or LT1 complex after X-galstaining. At a dose as low as 129 ng, LMD showed considerabletransfection activity, still higher than that of LD (DC-Chol:DOPEcomplexed to DNA, 3:1 w/w ratio) (DNA dose 5.0 μg). We found much higherreporter gene expression with LMD compared to transfection mediated byLD and liposome:LT1:DNA complexes. In fact, LMD mediated transfectionwas over 19 times more effective than LD and over 4 times more effectivethan liposome:LT1:DNA at comparable doses.

Example 13 Comparison with GL-67 Cationic Liposomes; In Vivo Experimentin Mouse Lung

[0212] We assessed the transfection activity in mouse lung in vivo ofLMD (prepared as described in Example 4 using DC-Chol:DOPE cationicliposomes [3:2, m/m] and pCF1-CAT plasmid), comparing this with thetransfection activity of cationic liposomes GL-67:DOPE:DMPE-PEG₅₀₀₀(1:2:0.05 m/m/m) complexed with pCF1-CAT plasmid (LD) (liposome:DNAratio 5.4:1 w/w) used to great effect in lung clinical trials (Alton etal., Lancet, 1999, 353, 947-954).

[0213] LMD (final DNA concentration 0.14 mg/ml; 100 μl volume; DNA dose14 μg) was instilled into the lungs of Balb/c mice.GL-67:DOPE:DMPE-PEG₅₀₀₀ (1:2:0.05 m/m/m) was complexed with pCF1-CATplasmid (final DNA concentration 0.8 mg/ml; 100 μl volume; DNA dose 80μg) and this LD complex was similarly instilled into the lungs of Balb/cmice. After 48 h, the lungs were homogenised and assayed for CATactivity. Error bars indicate s.e.m.

[0214] The results show (FIG. 18) that LMD and the GL-67 containing LDsystem gave essentially equivalent levels of transfection in vivo eventhough the LMD system was delivering a five fold lower DNA dose.

Example 14 Sugar Modified LMD Systems

[0215] Unspecific interactions of LMD with the biological environmentshould be minimised for in vivo applications. For example, duringintravenous administrations undesired interactions with blood components(salts, proteins . . . ) and non-target cells are important obstacles.This opsonization of foreign particles with plasma proteins presents oneof the first steps in the natural process of removal of foreignparticles by the innate immune system. To reduce proteins binding andsalt induced aggregations, naturally occurring polysaccharides can becoupled to LMD. This carbohydrate modification of LMD can be as wellapplied for targeting of LMD to carbohydrates receptors.

[0216] To obtain the desired effect, we designed the neoglycolipidsdescribed in FIG. 19. Those compounds are based onto three distinctdomains.

[0217] ACHx (CJE 52): This lipid (see FIG. 9) was chosen as genericlipid platform for the desired neoglycolipids. The cholesterol aliphaticring system represents a very hydrophobic area that inserts inside thelipid coat of LMD or LD particles acting as a neoglycolipid anchor.

[0218] Carbohydrate motif: The choice of oligosaccharides was limited bythe complexity of any chemistry involving carbohydrate modifications. Wedecided to use the long chain commercially available carbohydratesmaltotetraose and maltohepataose as proof of principle.

[0219] Linker: Use of a chemoselective linkage proved efficient andflexible, allowing us to synthesise a wide range of neoglycolipids. Thischemoselective technique was based upon a conversion of CJE52 into anhydroxylamino lipid that was able to couple directly to unprotectedcarbohydrates. The synthesis of a typical hydroxylamino-CJE52 is shownin FIG. 20—Scheme 1 and the coupling of the carbohydrate moiety onto thelinker is based on the glycosylation of an O-substituted hydroxylamine(The principle of the reaction with Glucose is illustrated in FIG.21—Scheme 2). Following this strategy, Maltotetraose and Maltoheptaosewere coupled to obtain GLU4 and GLU7 compounds (Structure in FIG. 22).

[0220] The glyco-modification of LMD was based on the natural ability ofneoglycolipid micelles to dissociate and free lipids incorporate intoLMD membranes. Firstly LMD were formulated from DC-Chol:DOPE cationicliposomes, Mu1 peptide and pCMVβ plasmid as described in Example 4.Thereafter, a suspension of neoglycolipid micelles in Hepes Buffer, pH7.0 was added to LMD mixtures and the whole incubated for 30 min at 20°C. before storage at −80° C. (FIG. 23).

[0221] Neoglycolipids Stabilisation of LMD:

[0222] The stabilisation effect of neoglycolipid modified LMD wasevaluated by incorporation of 7.5 mol % of GLU4 or GLU7 into LMD. Thelipid layer of LD systems is known to aggregate after salt exposure.Therefore, the sizes of LD (final DNA concentration 1 μg/ml) particleswere evaluated after 30 min at 37° C. in OPTIMEM by Photon CorrelationSpectroscopy (N4 plus, Coulter). Unimodal analysis was used to evaluatethe mean particle size. The average percentage increase in LD particlesize is shown (FIG. 24). The same procedure was followed for the basicLMD system, LMD(GLU4) and LMD(GLU7) (final DNA concentrations 1 μg/ml).

[0223] The results indicate that LMD is more stable than LD in solutionbut also show that the presence of GLU4 and GLU7 has an enhancedanti-aggregation stabilising effect on LMD particles at 7.5 mol %.

[0224] In vitro transfection efficiency: transfection activity wasdetermined with Hela cells seeded at 5×10⁴ cells per well in 24-wellculture plates and grown to approximately 70% confluence in DMEMsupplemented with FCS at 37° C. and in the presence of 5% CO₂. Cellswere washed in PBS and then treated with solutions containing LMDcomplexes, prediluted with DMEM containing FCS at the indicatedpercentages (%) (final DNA concentration 5.0 μg/ml in all cases), for 30min. Cells were further washed and then incubated for a further 48 h innormal medium (NGM) prior to harvesting. The level of β-galactosidaseexpression was determined with a standard assay kit (chemiluminescent,Roche).

[0225] The results indicate an enhancement of the transfectionefficiency due to Sugar modification in both 0% and 50% Serum conditions(FIG. 25).

[0226] Discussion

[0227] We have previously shown that DC-Chol/DOPE liposomes areefficient at transfecting the neuronally derived ND7 cell line ³¹.DC-Chol has been used successfully outside the CNS in a variety oftissues and has undergone clinical trials for gene therapy treatments ofcystic fibrosis ^(33, 34). Also, DC-Chol liposomes have been shown notto exhibit cytotoxic side effects ^(35, 36). For these reasons we wishto develop improved formulations of these liposomes for use in neuralcells.

[0228] We describe here the use of a virus-coded protein for cellulartransfection. We found that Mu1, when used in combination with thecationic liposome DC-Chol/DOPE was able to improve significantlycellular transfection. This effect was most likely due to the ability ofMu1 to condense pDNA and could be optimized by varying the ratios ofpolypeptide, pDNA and cationic liposome. Significantly, the enhancementin transfection efficiency was more pronounced on differentiated cells.As mentioned above, ND7 cells were derived from primary DRGs.Differentiating ND7 cells induces a phenotype similar to their parentalperipheral sensory neurons including the induction of neurite outgrowth,a reduction in overall proliferation and a reduction in transfectability^(28, 37). An enhancement in transfection efficiency in differentiatedND7s may reflect an enhanced ability to promote transfections in primaryneurons or in vivo.

[0229] The success of non-viral gene delivery vehicles as viablealternatives to virus vector-based systems is dependent on thedevelopment of complexes with higher and longer lasting transfectionefficiencies. Since the initial identification of cationic liposomes asvehicles for the transfer of genetic material into cells there has beena large push to develop better cationic liposome formulations ^(5, 7).Most attempts at improving cationic liposomes have been based onstructural modifications to the molecule itself ³⁰. Novel formulationshave been developed which have improved transfection efficiencies ³⁰.However, particular cell types behave differently in regards to cationicliposomal transfection. For example, we found the polypeptide Mu1 betterat enhancing cationic liposome mediated transfection than Vp1. This wasprobably due to Mu1's greater charge ratio. While both peptides areapproximately the same molecular weight, the overall charge ratio of Mu1was more than twice that of Vp1 (Table 1). Consistent with this Mu1 wasable to retard the electrophoretic mobility of plasmid DNA at less than{fraction (1/60)}^(th) the concentration demonstrating how tightly Mu1is able to bind DNA. While a small shift in pDNA mobility was detectedwhen 0.25 μg Mu1 was complexed to 1 μg pCMVβ, almost all of the plasmidwas retained near the loading well following addition of 0.5 μg Mu1(FIG. 1). A 0.5/1.0 (w/w) ratio of Mu1 to pCMVβ corresponds to a 1000/1molar ratio. Each molecule of Mu1 contains 12 residues that couldpotentially carry a positive charge. The theoretical charge ratio of Mu1to pCMVβ would then be 1.6 (12000 Mu1 cations to 7500 pCMVβ anions).This ratio should completely neutralize the negative charges on pCMVβthus completely retarding its migration as seen.

[0230] A direct comparison between the amount of Mu1 that significantlyretarded plasmid DNA migration and that which optimally enhancedtransfections could not be made since the method of preparation wasdifferent The peptide-pDNA-liposome transfection complexes were preparedin larger volumes (see Materials and Methods). Although it took 24 timesas much Mu1 (12 μg/1 μg pCMVβ) to achieve optimal enhancement oftransfection efficiencies as it did to retard migration in an agarosegel, the concentration in solution was similar (25 ng/mL, pDNAretardation; 30 ng/mL, optimal transfections). The presence of Mu1 alsoaltered cationic liposome pDNA interactions. The optimal ratio ofDC-Chol/DOPE to pCMVβ in the presence of Mu1 was 6/1, twice thatpreviously found optimal in neuronal cells ^(31, 38). Theoretically theamount of Mu1 used should have completely neutralized the positivecharges on pCMVβ, which would have prevented further complexing withDC-Chol/DOPE. Clearly this was not the case since much improvedtransfection efficiencies were attainable. It's likely that not all thepossible charged amino acids were protonated in our buffer conditions.Why more cationic liposomes are required to improve transfections is notclear and we are currently working to address this question.

[0231] Finally a point should be made regarding the nuclear localizationsignal embedded within Vp1. Recent evidence in our laboratory(unpublished observations) and in others ^(10, 11), ^(39, 40) hassuggested that nuclear transport of transfected material may beinefficient in lipofection. For this reason attempts have been made topre-condense DNA with polycations containing peptide sequences known tohave nuclear localizing capabilities with the aim of improving nuclearuptake of transfected DNA ^(17, 20, 22). We found however, that the moreefficient DNA condensing properties of Mu1 far outweighed the nuclearlocalizing capacity of Vp1 in terms of improving transfectionefficiencies. Similarly Fritz et al., ²² found no difference intransfection efficiencies between recombinant human histone (H1) and amodified version containing the SV40 large T antigen nuclear localizingsequence. Other studies have suggested that the presence of an NLS doesimprove nuclear accumulation of transfected pDNA albeit via specificintracellular pathways ^(41, 42).

[0232] All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

[0233] References

[0234] 1. Wood M J A et al. Inflammatory effects of gene-transfer intothe CNS with defective HSV-1 vectors. Gene Ther 1994; 1: 283-291.

[0235] 2. Byrnes A P et al. Adenovirus gene-transfer causes inflammationin the brain. Neuroscience 1995; 66: 1015-1024.

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1. A non-viral nucleic acid delivery vector comprising a condensedpolypeptide/nucleic acid complex and a cationic lipid, wherein thecomplex comprises (a) a nucleic acid sequence of interest (NOI); and (b)one or more viral nucleic acid packaging polypeptides, or derivativesthereof, said polypeptides or derivatives thereof being (i) capable ofbinding to the NOI; and (ii) capable of condensing the NOI; and whereinthe NOI is heterologous to the polypeptide.
 2. A vector according toclaim 1 wherein at least one polypeptide is an adenoviral nucleic acidpackaging polypeptide, or derivative thereof.
 3. A vector according toclaim 2 wherein the adenoviral polypeptide is Mu1, pV or pVII or aderivative thereof.
 4. A vector according to any one of claims 1 to 3further comprising a polypeptide comprising a nuclear localisationsequence (NLS).
 5. A vector according to claim 4 wherein the polypeptidecomprising a nuclear localisation sequence (NLS) is adenoviral pV or aderivative thereof.
 6. A condensed polypeptide/nucleic acid complexcomprising a cationic lipid, a polypeptide component and a nucleic acidcomponent, for use in delivering the nucleic acid component to a nucleusof a eukaryotic cell, wherein (i) the polypeptide component is a viralnucleic acid packaging polypeptide, or derivative thereof; (ii) thepolypeptide component or derivative thereof is capable of binding to theNOI; and (iii) the polypeptide component or derivative thereof iscapable of condensing the NOI; and wherein the nucleic acid isheterologous to the polypeptide.
 7. A complex according to claim 6wherein at least one polypeptide is an adenoviral nucleic acid packagingpolypeptide, or derivative thereof.
 8. A complex according to claim 7wherein the adenoviral polypeptide is Mu1, pV or pVII or a derivativethereof.
 9. A complex according to any one of claims 6 to 8 furthercomprising a polypeptide comprising a nuclear localisation sequence(NLS).
 10. A complex according to claim 9 wherein the polypeptidecomprising a nuclear localisation sequence (NLS) is adenoviral pV or aderivative thereof.
 11. A complex according to any one of claims 6 to 10wherein the ratio liposome:NOI:polypeptide is 2-20:1:0.5-1, preferably10-14:1:0.5-0.7, more preferably approximately 12:1:0.6.
 12. A method ofproducing a non-viral nucleic acid delivery vector comprising a cationiclipid and a condensed polypeptide/ nucleic acid complex, which methodcomprises (a) contacting an nucleic acid sequence of interest (NOI) witha viral nucleic acid packaging polypeptide or derivative thereof, saidpolypeptide component or derivative thereof being (i) capable of bindingto the NOI; and (ii) capable of condensing the NOI; and wherein the NOIis heterologous to the polypeptide; and (b) contacting the nucleicacid/polypeptide complex thusformed with a cationic lipid.
 13. A methodof introducing a nucleic acid sequence of interest (NOI) into aeukaryotic cell which method comprises contacting the cell with acomplex according to any one of claims 6 to 11, wherein the complexcomprises the NOI.
 14. A method according to claim 13 wherein the cellis a neuronal cell, cancer cell or epithelial cell.
 15. Use of a viralnucleic acid packaging polypeptide or derivative thereof in themanufacture of a nucleic acid delivery vector as defined in claim 1.